Fluorescent two-hybrid (f2h) assay for direct visualization of protein interactions in living cells

ABSTRACT

The present invention relates to an in vitro method for detecting protein-protein interactions comprising: (a) expressing in a eukaryotic cell a first fusion protein comprising (i) a (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell or interacts with proteinaceous or non-proteinaceous structures accumulated at distinct sites in the nucleus of the cell; and (ii) a (poly)peptide specifically binding to GFP; (b) expressing in the same cell a second fusion protein comprising (i) GFP; and (ii) a bait (poly)peptide; (c) expressing in the same cell a third fusion protein comprising (i) a fluorescent (poly)peptide, the excitation and/or emission wavelength of which differs from that of GFP; and (ii) a prey (poly)peptide; and (d) detecting the fluorescence emission of the fluorescent parts of the second and the third fusion protein in the cell upon excitation, wherein a co-localization of the fluorescence emission of both fusion proteins in the cell nucleus is indicative of an interaction of the bait and the prey (poly)peptide. The invention also relates to an in vitro method for detecting protein-protein interactions comprising: (a) expressing in a eukaryotic cell a first fusion protein comprising (i) a fluorescent (poly)peptide; (ii) a (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell; and (iii) a bait (poly)peptide (b) expressing in the same cell a second fusion protein comprising (i) a fluorescent (poly)peptide, the excitation and/or emission wavelength of which differs from that of the fluorescent (poly)peptide comprised in said first fusion protein; and (ii) a prey (poly)peptide and (c) detecting the fluorescence emission of the fluorescent parts of the first and the second fusion protein in the cell upon excitation, wherein a co-localization of the fluorescence emission of both fusion proteins in the cell nucleus is indicative of an interaction of the bait and the prey (poly)peptide. Furthermore, the present invention relates to methods for identifying a compound modulating the interaction of two (poly)peptides and methods of determining the relative strength of the interaction of two proteins with a third protein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 12/811,712 filed Jul. 6, 2010 which is a 35 U.S.C. §371 U.S. National Stage Entry of International Application No. PCT/EP09/00067 filed Jan. 8, 2009, which claims the benefit of priority of EP Application No. 08000297.5 filed Jan. 9, 2008, the contents of each of which are incorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing filed, entitled 2001-1000USCONSEQLIST, was created on Aug. 7, 2014 and is 13,464 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to an in vitro method for detecting protein-protein interactions comprising: (a) expressing in a eukaryotic cell a first fusion protein comprising (i) a (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell; and (ii) a (poly)peptide specifically binding to GFP; (b) expressing in the same cell a second fusion protein comprising (i) GFP; and (ii) a bait (poly)peptide; (c) expressing in the same cell a third fusion protein comprising (i) a fluorescent (poly)peptide, the excitation and/or emission wavelength of which differs from that of GFP; and (ii) a prey (poly)peptide; and (d) detecting the fluorescence emission of the fluorescent parts of the second and the third fusion protein in the cell upon excitation, wherein a co-localization of the fluorescence emission of both fusion proteins in the cell nucleus is indicative of an interaction of the bait and the prey (poly)peptide. The invention also relates to an in vitro method for detecting protein-protein interactions comprising: (a) expressing in a eukaryotic cell a first fusion protein comprising (i) a fluorescent (poly)peptide; (ii) a (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell; and (iii) a bait (poly)peptide; (b) expressing in the same cell a second fusion protein comprising (i) a fluorescent (poly)peptide, the excitation and/or emission wavelength of which differs from that of the fluorescent (poly)peptide comprised in said first fusion protein; and (ii) a prey (poly)peptide and (c) detecting the fluorescence emission of the fluorescent parts of the first and the second fusion protein in the cell upon excitation, wherein a co-localization of the fluorescence emission of both fusion proteins in the cell nucleus is indicative of an interaction of the bait and the prey (poly)peptide. Furthermore, the present invention relates to methods for identifying a compound modulating the interaction of two (poly)peptides and methods of determining the relative strength of the interaction of two proteins with a third protein.

BACKGROUND OF THE INVENTION

In this specification, a number of documents including patent applications and manufacturer's manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

After sequencing the human genome, the next challenge is now to analyze the complex protein-networks underlying cellular functions. In the last decade, a wide variety of methods to study protein-protein interactions ranging from biochemical to genetic or cell-based approaches have been developed. Biochemical methods such as affinity purification or co-immunoprecipitation (Co-IP) allow the detection of protein complexes in vitro. Genetic methods, such as the yeast two-hybrid (Y2H) system, enable efficient high-throughput screening of interactions within the cellular environment. The analysis of mammalian protein-protein interactions in yeast may, however, suffer from the absence or insufficient conservation of cellular factors modulating protein-protein interactions, e.g. through posttranslational modifications (Parrish et al., 2006). Furthermore, this method is laborious and error-prone.

In recent years new fluorescence-based methods for in-cell visualization of protein-protein-interactions have been introduced. Two established techniques, fluorescence resonance energy transfer (FRET) (Miyawaki, 2003; Sekar and Periasamy, 2003) and bimolecular fluorescence complementation (BiFC) (Kerppola, 2006), are based on the expression of fluorescently labeled proteins or fragments thereof. However, FRET requires costly instrumentation and advanced technical expertise, while BiFC is based on the irreversible complementation and slow maturation of fluorophores which does not allow real-time detection of protein-protein interactions (Kerppola, 2006).

All these methods have inherent shortcomings and are typically combined to obtain more reliable results.

Thus, there is a need for improved methods of detecting protein-protein interactions.

The solution to said technical problem is achieved by providing the embodiments characterized in the claims.

Accordingly, in a first aspect the present invention relates to an in vitro method for detecting protein-protein interactions comprising: (a) expressing in a eukaryotic cell a first fusion protein comprising (i) a (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell; and (ii) a (poly)peptide specifically binding to GFP; (b) expressing in the same cell a second fusion protein comprising (i) GFP; and (ii) a bait (poly)peptide; (c) expressing in the same cell a third fusion protein comprising (i) a fluorescent (poly)peptide, the excitation and/or emission wavelength of which differs from that of GFP; and (ii) a prey (poly)peptide; and (d) detecting the fluorescence emission of the fluorescent parts of the second and the third fusion protein in the cell upon excitation, wherein a co-localization of the fluorescence emission of both fusion proteins in the cell nucleus is indicative of an interaction of the bait and the prey (poly)peptide.

The term “protein-protein interactions” refers to the specific interaction of two or more proteinaceous compounds, i.e. poly(peptides) or proteins. Specific interaction is characterized by a minimum binding strength or affinity. Binding affinities for specific interactions generally reach from the pM to the mM range and also largely depend on the chemical environment, e.g. the pH value, the ionic strength, the presence of co-factors etc. In the context of the present invention, the term particularly refers to protein-protein interactions occurring under physiological conditions, i.e. in a cell.

The term “expressing in a eukaryotic cell” relates to the transcription and translation of the fusion proteins of the invention using appropriate expression control elements that function in the chosen cell. In this manner, the binding properties of individual fusion proteins may be tested in cellular expression systems. To this end, a nucleic acid molecule encoding a fusion protein may be cloned into a suitable expression vector, the composition of which generally depends on the expression system. For the present invention, the expression system is eukaryotic, preferably mammalian. A typical mammalian expression vector contains a promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements might include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from retroviruses, e.g., RSV, HTLVI, HIVI, and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter). Possible examples for regulatory elements ensuring the initiation of transcription comprise the cytomegalovirus (CMV) promoter, RSV-promoter (Rous sarcoma virus), the lacZ promoter, the gal10 promoter, human elongation factor 1a-promoter, CMV enhancer, CaM-kinase promoter, the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedral promoter or the SV40-enhancer. Examples for transcription termination signals are the SV40-poly-A site or the tk-poly-A site or the SV40, lacZ and AcMNPV polyhedral polyadenylation signals, downstream of the polynucleotide. Moreover, elements such as origin of replication, drug resistance genes, regulators (as part of an inducible promoter) or internal ribosomal entry sites (IRES) may also be included.

Suitable expression vectors for Drosophila are those belonging to the pMT DES system (Invitrogen) using the drosophila metallothionein (MT) promoter (Bunch et al., 1988) or pAC5.1 using the drosophila actin 5C promoter. A vector using the GAL4-inducible USA promoter is pUAST. Yeast vectors are the pYEp vector (using a Gal10 promoter), pYX142 (single copy vector) or pYX232 (2μ plasmid using the TPI triosephosphat isomerase promoter (both Novagen)).

Mammalian host cells that could be used include but are not restricted to human Hela, 293, H9, SH-EP1 and Jurkat cells, mouse NIH3T3 and C2C12 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells, Syrian golden baby hamster kidney (BHK) cells and Chinese hamster ovary (CHO) cells. Alternatively, the fusion proteins can be expressed in stable cell lines that contain the gene construct integrated into a chromosome. The co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells. The transfected nucleic acid molecule can also be amplified in the cell to express large amounts of the encoded fusion protein. The dhfr (dihydrofolate reductase) marker is useful to develop cell lines that carry several hundred or even several thousand copies of the gene of interest. Another useful selection marker is the enzyme glutamine synthase (GS) (Bebbington et al., 1992; Murphy et al., 1991). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. Appropriate culture media and conditions for the above-described host cells are known in the art.

The term “fusion protein” refers to chimeric proteins consisting of sequences derived from at least two different proteins or (poly)peptides. According to the teaching of the present invention, in exemplary fusion proteins, a bait (poly)peptide is fused to a fluorescent (poly)peptide and to a (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell. Alternatively, a prey (poly)peptide is fused to a fluorescent (poly)peptide. Fusion may be performed by any technique known to the skilled person, as long as it results in the in frame fusion of the nucleic acid molecules encoding the components of the fusion proteins of the invention. Fusion of the components may be effected in any order. Conventionally, the generation of a fusion protein from two or more separate (poly)peptides or domains is based on the “two-sided splicing by overlap extension” described in (Horton et al., 1989). The fragments coding for the single (poly)peptides are generated in two separate primary PCR reactions. The inner primers for the primary PCR reactions contain a significant, approximately 20 bp, complementary region that allows the fusion of the two domain fragments in the second PCR. Alternatively, the coding regions may be fused by making use of restriction sites which may either be naturally occurring or be introduced by recombinant DNA technology.

The term “(poly)peptide” as used herein describes a group of molecules which comprises the group of peptides, consisting of up to 30 amino acids, as well as the group of polypeptides, consisting of more than 30 amino acids. Also in line with the definition the term “(poly)peptide” describes fragments of proteins. (Poly)peptides may further form dimers, trimers and higher oligomers, i.e. consisting of more than one (poly)peptide molecule. (Poly)peptide molecules forming such dimers, trimers etc. may be identical or non-identical. The corresponding higher order structures are, consequently, termed homo- or heterodimers, homo- or heterotrimers etc. The terms “(poly)peptide” and “protein” also refer to naturally modified (poly)peptides/proteins wherein the modification is effected e.g. by glycosylation, acetylation, phosphorylation and the like. Such modifications are well known in the art.

The term “fluorescent (poly)peptide” or “fluorescent protein” refers to (poly)peptides emitting fluorescent light upon excitation at a specific wavelength. A variety of fluorescent proteins can be used in the present invention. One group of such fluorescent proteins includes Green Fluorescent Protein isolated from Aequorea victoria (GFP), as well as a number of GFP variants, such as cyan fluorescent protein, blue fluorescent protein, yellow fluorescent protein, etc. (Zhang et al., 2002; Zimmer, 2002). Typically, these variants share about 80%, or greater sequence identity with the amino acid sequence of SEQ ID No: 1 or the nucleic acid sequence of SEQ ID NO: 2, respectively. Color-shift GFP mutants have emission colors blue to yellow-green, increased brightness, and photostability (Tsien, 1998).

One such GFP mutant, termed the Enhanced Yellow Fluorescent Protein, displays an emission maximum at 529 nm. Additional GFP-based variants having modified excitation and emission spectra (Tsien et al., U.S. Patent Appn. 200201231 13A1), enhanced fluorescence intensity and thermal tolerance (Thastrup et al., U.S. Patent Appn. 20020107362A1; Bjorn et al., U.S. Patent Appn. 20020177189A1), and chromophore formation under reduced oxygen levels (Fisher, U.S. Pat. No. 6,414,119) have also been described.

Another group of fluorescent proteins includes the fluorescent proteins isolated from anthozoans, including without limitation the red fluorescent protein isolated from Discosoma species of coral, DsRed (Matz et al., 1999), e.g., the amino acid sequence of SEQ ID NO: 3 or the nucleic acid sequence of SEQ ID NO: 4, respectively (see, e.g., accession number AF168419). DsRed and the other anthozoan fluorescent proteins share only about 26-30% amino acid sequence identity to the wild-type GFP from Aequorea victoria, yet all the crucial motifs are conserved, indicating the formation of the 11-stranded beta-barrel structure characteristic of GFP. The crystal structure of DsRed has also been solved, and shows conservation of the 11-stranded beta-barrel structure of GFP (MMDB Id: 5742).

A number of mutants of the longer wavelength red fluorescent protein DsRed have also been described, and similarly, may be employed in the generation of the fusion proteins of the invention comprising fluorescent (poly)peptides. For example, recently described DsRed mutants with emission spectra shifted further to the red may be employed in the practice of the invention (Baird et al., 2000; Terskikh et al., 2000; Wiehler et al., 2001). Monomeric versions of Ds Red are e.g. mRFP (e.g. having the amino acid sequence of SEQ ID NO: 5 or the nucleic acid sequence of SEQ ID NO: 6), mRFP1 (Campbell et al., 2002), mCherry, mOrange or mPlum (Shaner et al., 2004) or TagRFP (Merzlyak et al., 2007) Most recently, GFPs from the anthozoans Renilla reniformis and Renilla kollikeri were described (Ward et al., U.S. Patent Appn. 20030013849).

An increasingly large number of other fluorescent proteins from a number of ocean life forms have recently been described, and the Protein Data Bank currently lists a number of GFP and GFP mutant crystal structures, as well as the crystal structures of various GFP analogs. Related fluorescent proteins with structures inferred to be similar to GFP from corals, sea pens, sea squirts, and sea anemones have been described, and may be used in the generation of the fusion proteins of the invention comprising fluorescent (poly)peptides (for reviews, see (Zhang et al., 2002; Zimmer, 2002)).

Fluorescent proteins from Anemonia majano, Zoanthus sp., Discosoma striata, Discosoma sp. and Clavularia sp. have also been reported (Matz et al., 1999). A fluorescent protein cloned from the stony coral species, Trachyphyllia geoffroyi, has been reported to emit green, yellow, and red light, and to convert from green light to red light emission upon exposure to UV light (Ando et al., 2002). Recently described fluorescent proteins from sea anemones include green and orange fluorescent proteins cloned from Anemonia sulcata (Wiedenmann et al., 2000), a naturally enhanced green fluorescent protein cloned from the tentacles of Heteractis magnifica (Tu et al., 2003), a generally non fluorescent purple chromoprotein displaying weak red fluorescence cloned from Anemonia sulcata and a mutant thereof displaying far-red shift emission spectra (595 nm) (Lukyanov et al., 2000). Additionally, another class of GFP-related proteins having chromophoric and fluorescent properties has been described. One such group of coral-derived proteins, the pocilloporins, exhibit a broad range of spectral and fluorescent characteristics (Dove and Hoegh-Guldberg, 1999, PCT application WO 00146233; (Dove et al., 2001)). Recently, the purification and crystallization of the pocilloporin Rtms5 from the reef-building coral Montipora efflorescens has been described (Beddoe et al., 2003). Rtms5 is deep blue in colour, yet is weakly fluorescent. However, it has been reported that Rtms5, as well as other chromoproteins with sequence homology to Rtms5, can be interconverted to a far-red fluorescent protein via single amino acid substitutions (Beddoe et al., 2003; Bulina et al., 2002; Lukyanov et al., 2000).

Various other coral-derived chromoproteins closely related to the pocilloporins are also known (see, for example, Gurskaya et al., 2001; Lukyanov et al., 2000). Further examples of fluorescent proteins are GFP form Renilla reniformis, mKO from Fungia concinna, Azami Green from Galaxeidae or cOFP from Cerianthus. Any of the fluorescent or chromophoric proteins or fluorescent or chromophoric fragments thereof may be used in accordance with the teaching of the present invention. Fragments of the fluorescent or chromophoric protein are preferably functional fragments.

Accumulation of a (poly)peptide at distinct sites in the nucleus of the cell may be caused by the (poly)peptide interacting with proteinaceous or non-proteinaceous structures already accumulated at distinct sites in the nucleus of the cell or by accumulating at a distinct site providing a suitable environment for the accumulation of the (poly)peptide, preferably by directly or indirectly binding to said sites.

“Co-localization of the fluorescence emission in the nucleus” denotes the localization of two different fluorescence emissions at the same site of the cell nucleus. Co-localization is detected as soon as two proteins interact with each other. Co-localization is detected as the partial or complete spatial overlap of fluorescence emission from two different fluorescent (poly)peptides in the nucleus. Detection can be effected by the experimenter or by specialized software known to the skilled person (e.g. ImageJ co-localization plug-ins, http://rsb.info.nih.gov/ij/).

The present invention relies on the development of a novel fluorescent two-hybrid (F2H) assay for the direct visualization of protein-protein interactions in living eukaryotic cells. The simple optical readout of this F2H assay allows observation of protein-protein interactions in real time and may be employed for high-throughput screens. The method of the invention is based on the immobilization of a fluorescently labeled bait (poly)peptide at a distinct nuclear structure enabling the detection of protein-protein interactions as co-localization of a differently labeled prey (poly)peptide at this defined structure. The F2H assay of the invention was tested on the example of cell lines with a stable integration of a lac operator array to immobilize a lac repressor fused to fluorescently labeled (poly)peptides of interest (bait (poly)peptides). Readily usable cell lines have already been described for human, mouse, hamster and Drosophila (Dietzel et al., 2004; Janicki et al., 2004; Robinett et al., 1996; Tsukamoto et al., 2000; Tumbar et al., 1999; Vazquez et al., 2001). To be independent of specific transgenic cell lines, this assay could be modified by using various cellular structures like the lamina or centrosomes as anchoring structures to locally immobilize bait (poly)peptides.

Like other genetic two-hybrid methods also the F2H assay of the invention may yield false positive or false negative results, which need to be controlled for. Prey (poly)peptides that bind to the lac operator array in the absence of a bait (poly)peptide can be identified by an initial screen. To this end the localization of prey (poly)peptides within the nucleus is determined by fluorescent microscopy in the absence of a respective bait protein. [0004]

Subsequently, a random accumulation of the fluorescent prey protein at the lac operator array can be determined by a clustered fluorescence at this structure. (Poly)peptides identified in this way can only be used as bait (poly)peptides to avoid false positive results. More than 20 protein-protein interactions from different subcellular compartments were analyzed with the F2H assay of the present invention and identical results as previously described with other genetic or biochemical methods were obtained. Proteins found to bind by themselves to the lac operator array (such as e.g. SUMO3 discussed in the examples) can only be used as a bait (poly)peptide. The results disclosed in the examples show that the F2H assay of the present invention is a reliable and broadly applicable method to study protein-protein interactions.

In some cases, proteins may accumulate at subnuclear foci and thus complicate the F2H analysis. To bypass this problem, the lac operator array could be visualized and identified with a third fluorescent fusion protein like CFP-LacI.

The present invention is further characterized in that a minimal construct or fusion protein comprising a (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell and a (poly)peptide specifically binding to GFP can serve for the rapid and efficient high-throughput screening of protein interactions. Upon co-expression of a second fusion protein comprising GFP and a bait (poly)peptide, the interaction of GFP with the GFP-binding (poly)peptide bound to the (poly)peptide accumulated at distinct sites in the nucleus of the cell is established thus constituting the bait complex. Upon co-expression of a further (third) fusion protein comprising a fluorescent (poly)peptide and a prey (poly)peptide, both fluorescent (poly)peptides co-localize upon interaction of the bait and prey (poly)peptides fused thereto.

Libraries of proteins fused to GFP and of nucleic acids encoding the latter have by now been designed and established, (Newman et al., 2006), which greatly facilitates high-throughput screening for interaction partners of a protein.

In the international patent application WO 2007/068313, the present inventors disclose a (poly)peptide specifically binding to GFP derived from a camel VHH domain.

Accordingly, it is preferred that the protein specifically binding to GFP comprises the amino acid sequence as shown in SEQ ID NO: 7 or 9 or encoded by a nucleic acid molecule comprising a sequence as shown in SEQ ID NO: 8 or 10.

In summary, this new F2H assay allows the direct visualization of protein-protein interactions and should be ideally suited to investigate cell cycle or differentiation dependent changes in real-time in living cells. A significant advantage of the F2H assay over other cell-based techniques is its simplicity that does neither require costly instrumentation nor advanced technical expertise. The simple optical read-out of the F2H assay additionally offers the possibility to use this assay in automated high-throughput screens to systematically analyze the protein interactome in living cells.

As compared to the method described in (Miller et al., 2007) which relies on viral structures randomly accumulating in the cytoplasm and forming irregular and unforeseeable structures, the nuclear structures used in the present invention form defined and identifiable spots. In contrast, the method of Miller et al. aims at and results in many poorly defined and large cytoplasmic aggregates which are difficult to distinguish from unspecific aggregates often observed with artificially over-expressed fusion proteins or cytoplasmic vesicles. Using inert structures such as the lac-operator, it is immediately possible to detect unspecific aggregation of a fluorescent (poly)peptide, e.g. by the presence of a varying number of aggregates in the cells. In the method of the present invention, depending on the ploidy of the cell one to two spots are detectable.

In a second aspect, the present invention relates to an in vitro method for identifying a compound modulating the interaction of two (poly)peptides (a) expressing in a eukaryotic cell a first fusion protein comprising (i) a (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell and (ii) a (poly)peptide specifically binding to GFP; (b) expressing in the same cell a second fusion protein comprising (i) GFP; and (ii) a bait (poly)peptide; (c) expressing in the same cell a third fusion protein comprising (i) a fluorescent (poly)peptide, the excitation and/or emission wavelength of which differs from that of GFP and (ii) a prey (poly)peptide known or suspected to interact with the bait (poly)peptide; (d) contacting the cell with a test compound; and (e) detecting the fluorescence emission of the fluorescent parts of the second and the third fusion protein in the cell upon excitation; wherein a change in the degree of co-localization of the fluorescence emission of the fluorescent parts of the second and the third fusion protein in the cell nucleus as compared to that observed in the nucleus of a reference cell not contacted with the test compound is indicative that the compound is capable of modulating the interaction of the bait and the prey (poly)peptide.

“Modulating the interaction of two (poly)peptides” denotes the capability of certain compounds to influence the interaction of two (poly)peptides or proteins. The interaction can either be strengthened or weakened up to its complete abrogation. Modulation in the form of a weakening can take place e.g. by competition of a test compound with the interacting protein for the same binding site or by binding to one protein and thus altering its three dimensional structure so that the interaction between the two proteins is no longer possible due to conformational changes in said protein. On the other hand, the latter can also lead to an increase in the binding affinity towards a protein for the same reason.

A test compound can be but is not restricted to a compound belonging to the classes of e.g. nucleic acids, (poly)peptides, peptide aptamers, nucleic acid based aptamers, small molecules or antibodies or fragments thereof. The test compound can be any chemical compound.

Nucleic acids can be DNA, RNA or ribozymes. Nucleic acids can be synthesized chemically or produced in conjunction with a promoter by biological expression in vitro or even in vivo.

Nucleic acids can be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Suppliers of RNA synthesis reagents are Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, Ill., USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA), and Cruachem (Glasgow, UK).

A ribozyme (from ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA) is an RNA molecule that catalyzes a chemical reaction. Many natural ribozymes catalyze either their own cleavage or the cleavage of other RNAs, but they have also been found to catalyze the aminotransferase activity of the ribosome.

Examples of well-characterized small self-cleaving RNAs are the hammerhead, hairpin, hepatitis delta virus, and in vitro-selected lead-dependent ribozymes. The organization of these small catalysts is contrasted to that of larger ribozymes, such as the group I intron.

Aptamers are oligonucleic acid or peptide molecules that bind a specific target molecule. Aptamers are usually created by selecting them from a large random sequence pool, but natural aptamers also exist in riboswitches. Aptamers can be used for both basic research and clinical purposes as macromolecular drugs. Aptamers can be combined with ribozymes to self-cleave in the presence of their target molecule. These compound molecules have additional research, industrial and clinical applications. More specifically, aptamers can be classified as DNA or RNA aptamers or peptide aptamers. Whereas the former consist of (usually short) strands of oligonucleotides, the latter consist of a short variable peptide domain, attached at both ends to a protein scaffold.

Nucleic acid aptamers are nucleic acid species that have been engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms.

Peptide aptamers are (poly)peptides that are designed to interfere with other protein interactions inside cells. They consist of a variable peptide loop attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to that of an antibody (nanomolar range). The variable loop length is typically comprised of 10 to 20 amino acids, and the scaffold may be any protein, which has good solubility properties.

Currently, the bacterial protein Thioredoxin-A is the most frequently used scaffold protein, the variable loop being inserted within the reducing active site, which is a -Cys-Gly-Pro-Cys-loop in the wild protein, the two cystein lateral chains being able to form a disulfide bridge. Peptide aptamer selection can be made using different systems, but the most frequently used one is currently the yeast two-hybrid system.

An antibody can be, for example, polyclonal or monoclonal. The term “antibody” also comprises derivatives or fragments thereof which still retain the binding specificity. Techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane “Antibodies, A Laboratory Manual”, Cold Spring Harbor Laboratory Press, 1988 and Harlow and Lane “Using Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, 1999.

An antibody also includes chimeric, single chain and humanized antibodies, as well as antibody fragments, like, inter alia, Fab fragments. Antibody fragments or derivatives further comprise F(ab′)2, Fv or scFv fragments; see, for example, Harlow and Lane (1988) and (1999), loc. cit.

A small molecule according to the present invention can be organic or inorganic and has a molecular weight of up to 2000 Daltons, preferably not more than 1000 Daltons, most preferably not more than 800 Daltons.

In a preferred embodiment of this aspect of the present invention, the test compound is capable of weakening the interaction of two (poly)peptides, wherein a decrease of co-localization of the fluorescent parts of both fusion proteins in the cell nucleus as compared to that observed in the nucleus of a reference cell not contacted with the test compound is indicative of the compound being capable of weakening the interaction of the bait and the prey (poly)peptide.

“Capable of weakening the interaction of two (poly)peptides” in context with the present invention means firstly the influence of a compound on the binding affinity of a (poly)peptide towards another (poly)peptide leading to a weakening or complete disruption/abrogation of the interaction or binding affinity. As described above, this may take place by conformational changes induced in one (poly)peptide upon binding of the compound. Secondly, the compound may directly compete with the interacting (poly)peptide for the same or a different binding site adjacent to said binding site on the other (poly)peptide. A weakening or complete abrogation of interaction is detectable as lessening or complete absence of co-localization of both fluorescent signals.

A reference cell in the context of the present invention denotes a cell that has not been contacted with the test compound. Accordingly, the influence of the compound on the interaction behaviour of the bait and prey (poly)peptide can be compared directly.

In another preferred embodiment of this aspect of the invention, the test compound is capable of inducing or enhancing the interaction of two (poly)peptides, wherein an increase in co-localization of the fluorescent parts of both fusion proteins in the cell nucleus as compared to that observed in the nucleus of a reference cell not contacted with the test compound is indicative of the compound being capable of inducing or enhancing the interaction of the bait and the prey (poly)peptide.

“Capable of inducing the interaction of two (poly)peptides” denotes the influence of a compound on the binding affinity of a (poly)peptide towards another (poly)peptide leading to an increased binding affinity. For example, if no or only minor co-localization of the fluorescent signals of the bait and the prey (poly)peptide are detected in the reference cell, an increase in co-localization indicates a strengthened interaction.

In a different aspect, the present invention relates to an in vitro method of determining the relative strength of the interaction of two proteins with a third protein comprising: (a) expressing in a eukaryotic cell a first fusion protein comprising (i) a (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell; and (ii) a (poly)peptide specifically binding to GFP; (b) expressing in the same cell a second fusion protein comprising (i) GFP; and (ii) a bait (poly)peptide; (c) expressing in the same cell a third fusion protein comprising (i) a fluorescent (poly)peptide, the excitation and/or emission wavelength of which differs from that of GFP; and (ii) a first prey (poly)peptide (d) expressing in the same cell a fourth fusion protein comprising (i) a fluorescent (poly)peptide, the excitation and/or emission wavelength of which differs from that of GFP and from that of the fluorescent (poly)peptide of said second fusion protein; and (ii) a second prey (poly)peptide; and (e) detecting the fluorescence emission of the fluorescent parts of the second and the third fusion protein in the cell upon excitation, wherein an extent of co-localization of the fluorescence emission of the second and the third fusion protein different as compared to that of the second and the fourth fusion protein in the cell nucleus is indicative of a different binding strength of the first and the second prey (poly)peptide to the bait (poly)peptide.

This and the following two aspects of the invention rely on two basic constructs, a prey and a bait construct, and can be applied in cases where libraries of nucleic acids encoding proteins comprising GFP are not available. The constructs may be contained in two separate vectors or in one vector.

In a further aspect, the present invention relates to an in vitro method for detecting protein-protein interactions comprising: (a) expressing in a eukaryotic cell a first fusion protein comprising (i) a fluorescent (poly)peptide; (ii) a (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell; and (iii) a bait (poly)peptide; (b) expressing in the same cell a second fusion protein comprising (i) a fluorescent (poly)peptide, the excitation and/or emission wavelength of which differs from that of the fluorescent (poly)peptide comprised in said first fusion protein; and (ii) a prey (poly)peptide and (c) detecting the fluorescence emission of the fluorescent parts of the first and the second fusion protein in the cell upon excitation, wherein a co-localization of the fluorescence emission of both fusion proteins in the cell nucleus is indicative of an interaction of the bait and the prey (poly)peptide.

In a different aspect, the present invention relates to an in vitro method for identifying a compound modulating the interaction of two (poly)peptides (a) expressing in a eukaryotic cell a first fusion protein comprising (i) a fluorescent (poly)peptide; (ii) a (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell; and (iii) a bait (poly)peptide; (b) expressing in the same cell a second fusion protein comprising (i) a fluorescent (poly)peptide, the excitation and/or emission wavelength of which differs from that of the fluorescent (poly)peptide comprised in said first fusion protein; and (ii) a prey (poly)peptide known or suspected to interact with the bait (poly)peptide; (c) contacting the cell with a test compound; and (d) detecting the fluorescence emission of the fluorescent parts of the first and the second fusion protein in the cell upon excitation; wherein a change in the degree of co-localization of the fluorescence emission of the fluorescent parts of both fusion proteins in the cell nucleus as compared to that observed in the nucleus of a reference cell not contacted with the test compound is indicative that the compound is capable of modulating the interaction of the bait and the prey (poly)peptide.

In a further aspect, the present invention relates to a method of determining the relative strength of the interaction of two proteins (interchangeably used with the term “(poly)peptides”) with a third protein (or (poly)peptide) comprising (a) expressing in a eukaryotic cell a first fusion protein comprising (i) a fluorescent (poly)peptide; (ii) a (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell; and (iii) a bait (poly)peptide; (b) expressing in the same cell a second fusion protein comprising (i) a fluorescent (poly)peptide, the excitation and/or emission wavelength of which differs from that of the fluorescent (poly)peptide comprised in said first fusion protein; and (ii) a first prey (poly)peptide; (c) expressing in the same cell a third fusion protein comprising (i) a fluorescent (poly)peptide, the excitation and/or emission wavelength of which differs from that of the fluorescent (poly)peptide comprised in said first and second fusion protein; and (ii) a second prey (poly)peptide; and (d) detecting the fluorescence emission of the fluorescent parts of the first, the second and the third fusion protein in the cell upon excitation, wherein an extent of co-localization of the fluorescence emission of the first and the second fusion protein different as compared to that of the first and the third fusion protein in the cell nucleus is indicative of a different binding strength of the first and the second prey protein to the bait (poly)peptide.

In a preferred embodiment of the aspects of the present invention relating to methods for detecting protein-protein interactions, the detection is employed for investigating the dependency of protein-protein interactions on cellular processes, wherein the method further comprises (d1) monitoring the fluorescence emission of the fluorescent parts of the first and the second fusion protein in the cell in the course of one or more processes in the cell; wherein a change in the degree of co-localization of the fluorescence emission of the fluorescent parts of both fusion proteins in the cell is indicative of a dependency of the interaction on the one or more cellular processes.

“Dependency on cellular processes” denotes the possibility that the bait or prey (poly)peptide or both alter their interaction behaviour due to modifications in the course of cellular processes such as the cell cycle. Proteins playing a role in the cell cycle undergo various modifications such as phosphorylation and dephosphorylation which might alter their binding affinity towards other proteins. Depending on the time point or period during a cellular process when this modification is effected, a (poly)peptide might loose or gain binding affinity to one or more other (poly)peptide.

In a more preferred embodiment, the cellular process is the cell cycle, secretion, translocation or signal transduction.

In another preferred embodiment, the detection is employed for determining the strength of a protein-protein interaction, wherein the method further comprises (d2) in case that a co-localization of the fluorescence emission of the fluorescent parts of both fusion proteins is detected, selective extinction of the fluorescence of said second fusion protein and monitoring the restoration of co-localization of the fluorescence emission of the fluorescent parts of both fusion proteins over time, wherein the time needed to establish co-localization is indicative of the strength of the protein-protein interaction.

This embodiment of the present invention in part utilizes the well-established FRAP (fluorescence recovery after photobleaching) method. This method denotes an optical technique capable of quantifying the diffusion and mobility of fluorescently labelled probes. This technique provides a great utility in biological studies of protein binding and is commonly used in conjunction with fluorescent proteins (FP), where the studied protein is fused to an FP. When excited by a specific wavelength of light (typically with a laser beam), the protein will fluoresce. When the protein that is being studied is produced with the FP, then the fluorescence can be tracked. After photodestruction of the FP (typically with a strong/intense laser pulse), the kinetic of fluorescence recovery in the bleached area provides information about strength of protein interactions, organelle continuity and protein trafficking that prevents or slows down the exchange of bleached and unbleached FPs. This observation has most recently been exploited to investigate protein binding.

In another preferred embodiment of the present invention, components (i), (ii) and/or (iii) of said first fusion protein and/or components (i) and (ii) of said second fusion protein and/or components (i) and (ii) of said third fusion protein are connected via a linker.

The term “linker” refers to the connection between the components of the fusion proteins of the invention. A linker can be a peptide bond or a stretch of amino acids comprising at least one amino acid residue which may be arranged between the components of the fusion proteins in any order. Such a linker may in some cases be useful, for example, to improve separate folding of the individual domains or to modulate the stability of the fusion protein. Moreover, such linker residues may contain signals for transport, protease recognition sequences or signals for secondary modification. The amino acid residues forming the linker may be structured or unstructured. Preferably, the linker may be as short as 1 amino acid residue or up to 2, 3, 4, 5, 10, 20 or 50 residues. In particular cases, the linker may even involve up to 100 or 150 residues.

In another preferred embodiment of the present invention, the (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell directly interacts with proteinaceous or non-proteinaceous structures accumulated at distinct sites in the nucleus of the cell.

In a different preferred embodiment of the present invention, the (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell indirectly interacts with proteinaceous or non-proteinaceous structures accumulated at distinct sites in the nucleus of the cell.

Indirect interaction may again occur via proteinaceous or non-proteinaceous molecules such as (poly)peptides or nucleic acids.

In a different preferred embodiment any of the prey (poly)peptide comprises a nuclear localization signal.

A nuclear localization signal (NLS) targets an expression product to the cell nucleus. An example of an NLS is the peptide sequence PKKKRKV of the SV40 large T-antigen, (Kalderon et al., 1984) which is capable of directing heterologous proteins into the nucleus. Further NLS are for example KR[PAATKKAGQA]KKKK, the NLS of nucleoplasmin as a prototype of an ubiquitous bipartite signal or KIPIK, the NLS of the yeast transcription repressor Matα2. Many other sequences of nuclear localization signals are known to the skilled person and described in the literature.

An NLS needs to be present if the prey (poly)peptide fused to the fluorescent (poly)peptide is per se not able to translocate to the nucleus. Generally, (poly)peptides of a size of up to 60 kDa may translocate to the nucleus. Furthermore, the amino acid composition of the (poly)peptide plays a role, i.e. a number of consecutive basic amino acids in a (poly)peptide may promote translocation of the (poly)peptide into the nucleus. In order to ensure that the prey (poly)peptide is translocated into and accumulated in the nucleus, it is preferred that an NLS is present. The same holds true for (poly)peptides accumulating at distinct sites of the nucleus but which only translocate to the nucleus if an NLS is present. An example for such a (poly)peptide is the lac repressor which by itself does not comprise an NLS.

In a different preferred embodiment, expression in the eukaryotic cell is effected by transfecting the nucleic acid molecules encoding said first and second fusion protein in one or more vectors.

Transfection is the introduction of nucleic acid molecules into eukaryotic cells. Commonly used methods comprise but are not restricted to transfection using calcium chloride, JetPEI™ (PolyPlus) or Lipofectin™ (Invitrogen).

The vector comprising the nucleic acid molecule encoding said first and/or second and/or third and/or fourth fusion protein is a eukaryotic expression vector, preferably a mammalian expression vector. Incorporation of the nucleic acid molecule into a vector offers the possibility of introducing the nucleic acid molecule efficiently into the cells and preferably the DNA of a host cell. The host cell may be a single cell such as a cell from a cell line. Such a measure renders it possible to express, if expression vectors are chosen, the respective nucleic acid molecule in the host cell. Thus, incorporation of the nucleic acid molecule into an expression vector opens up the way to a permanently elevated level of the encoded (poly)peptide or protein in any cell or a subset of selected cells of the host cell.

In another preferred embodiment, the (poly)peptide accumulated at distinct sites of the nucleus of the cell has been introduced into the cell. In a different preferred embodiment in case said (poly)peptide interacts with proteinaceous or non-proteinaceous structures accumulated at distinct sites in the nucleus, said proteinaceous or non-proteinaceous structures can be introduced into the cell.

Similarly to the vector(s) comprising the nucleic acid molecules encoding the fusion proteins of the present invention, also the nucleic acid molecules encoding the proteinaceous or non-proteinaceous structures can be introduced in a vector. The nucleic acid molecule is preferably stably integrated into the chromosomes of the cell leading to the generation of a stable cell line.

In a different preferred embodiment, the distinct sites of the cell form inert structures.

In a more preferred embodiment, the inert structure is the nuclear lamina or nuclear speckles.

In a different preferred embodiment, the (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell interacts with PML bodies (promyelotic leukemia; also termed PML nuclear bodies or PML NBs).

PML bodies have been associated with many nuclear functions including transcription, DNA repair, viral defence, stress, cell cycle regulation, proteolysis and apoptosis. The average mammalian cell contains 10-30 PML nuclear bodies. PML bodies are defined by the presence of the PML protein, first identified by its fusion to the retinoic acid receptor alpha in chromosomal translocation t(15,17) (Borden, 2002; Maul et al., 2000; Moller et al., 2003) associated with acute promyelocytic leukaemia (APL). PML protein is essential for the formation of PML NBs, and when it is absent, or its RING (comprising C₃HC₄ zinc finger the as a structural motif) fingers mutated, PML bodies are disrupted.

In a different preferred embodiment, the (poly)peptide accumulating at distinct sites of the nucleus or the proteinaceous or non-proteinaceous structures accumulated at distinct sites of the cell is heterologous.

“Heterologous” as used in the present invention denotes the origin of a proteinaceous or non-proteinaceous structure which is different from that of the cell in which it is expressed, i.e. a different species.

In another preferred embodiment, the (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell interacts with DNA.

In a more preferred embodiment, the DNA is the lac-operator.

In an even more preferred embodiment, the lac operator is present in the nucleus in multiple copies. The number of copies depends on the individual experiment, i.e. on the kind of host cell used or the bait (poly)peptide and/or the prey (poly)peptide. Commonly applied copy number reach from at least 150 copies of a plasmid comprising 256 copies of the lac operator (i.e. 38400 copies of the lac operator) to about 2000 copies of said plasmid (i.e. 512000 copies of the lac operator). In case a higher sensitivity is needed, the copy number can be even higher and reach up to 3000 copies. Alternatively, any kind of plasmid comprising a different copy number can be utilized to obtain the desired copy numbers of the lac operator. The lac operator can be arranged in tandem with or without nucleic acid stretches separating each element. The elements can be arranged head to tail or head to head. Cell lines having multiple copies of the lac operator stably integrated in the nucleus are known in the art (Janicki et al., 2004; Tsukamoto et al., 2000).

In another preferred embodiment, said first fusion protein comprises LacI.

The lac repressor Lad is a tetramer of identical subunits. Each subunit contains a helix-turn-helix (HTH) motif capable of binding to DNA. The operator site where the repressor binds is a DNA sequence with inverted repeat symmetry. The two DNA half-sites of the operator bind to two of the subunits of the tetrameric repressor.

Both the lac operator and Lad form part of the bacterial lac-operon, a regulatory unit for lactose metabolism of bacteria. If lactose is missing from the growth medium, the repressor binds very tightly to the lac operator located downstream of the promoter.

The present invention makes use of the interaction between Lad and the lac operator in order to form a non-proteinaceous anchor structure in the nucleus with which the method of the invention can be conveniently carried out. Generally, the interaction of Lad with the lac operator is detected at one distinct site in the nucleus.

An important advantage of this embodiment of the present invention as compared to previously known methods, in particular that of (Miller et al., 2007) is that the copy number of the lac operator forming a lac operator array can be varied in order to adapt the system to the expression level of both the bait and the prey (poly)peptides. For example, if the expression of one or both (poly)peptides is found too high in the cell, thus e.g. interfering with cellular processes or disturbing the fluorescent signal, another cell having a lower copy number of the lac operator can be taken, thus enabling for a titration of the detection of the interaction.

In a more preferred embodiment, said second fusion protein comprises GFP.

In another preferred embodiment, the detection is carried out using a fluorescence microscope.

A fluorescence microscope is a light microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption. The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, causing them to emit longer wavelengths of light (of a different color than the absorbed light). The illumination light is separated from the much weaker emitted fluorescence through the use of an emission filter. Typical components of a fluorescence microscope are the light source (Xenon or Mercury arc-discharge lamp), the excitation filter, the dichroic mirror (or dichromatic beamsplitter), and the emission filter. The filters and the dichroic mirror are chosen to match the spectral excitation and emission characteristics of the fluorophore used to label the specimen. Most fluorescence microscopes in use are epi-fluorescence microscopes (i.e.: excitation and observation of the fluorescence are from above (epi) the specimen). These microscopes have become an important part in the field of biology, opening the doors for more advanced microscope designs, such as the confocal laser scanning microscope (CLSM) and the total internal reflection fluorescence microscope (TIRF). These technologies are well known to the skilled person.

In another preferred embodiment, the eukaryotic cell is a living cell.

In another preferred embodiment, the method of the invention further comprises localizing the bait and/or prey (poly)peptides in the cell.

This embodiment serves to confirm that the co-localization effectively takes place in the nucleus and to rule out unspecific interaction of the bait and prey (poly)peptides. Localization is either detected by the experimenter or by specialized software able to distinguish different cellular compartments and well-known to the skilled person (e.g. ImageJ (Version 1.38, http://rsb.info.nih.gov/ij/).

In a different aspect, the present invention relates to a nucleic acid molecule encoding a fusion protein comprising (i) a fluorescent (poly)peptide; (ii) a (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell: and (iii) a bait (poly)peptide.

In another aspect, the present invention relates to a nucleic acid molecule encoding a fusion protein comprising (i) a (poly)peptide that binds to the lac-operator; and (ii) a (poly)peptide specifically binding to GFP, a vector comprising said nucleic acid and a protein encoded by said nucleic acid.

A “nucleic acid molecule”, in accordance with this aspect of the present invention, includes DNA, such as cDNA or genomic DNA, and RNA, both sense and anti-sense strands. Further included are nucleic acid mimicking molecules known in the art such as synthetic or semi-synthetic derivatives of DNA or RNA and mixed polymers. Such nucleic acid mimicking molecules or nucleic acid derivatives according to the invention include phosphorothioate nucleic acid, phosphoramidate nucleic acid, 2′-O-methoxyethyl ribonucleic acid, morpholino nucleic acid, hexitol nucleic acid (HNA) and locked nucleic acid (LNA) (see (Braasch and Corey, 2001)). LNA is an RNA derivative in which the ribose ring is constrained by a methylene linkage between the 2′-oxygen and the 4′-carbon. They may contain additional non-natural or derivative nucleotide bases, as will be readily appreciated by those skilled in the art.

In a preferred embodiment of this aspect of the invention, the fusion protein comprises LacI.

In another aspect, the present invention relates to a protein encoded by the nucleic acid molecule of the invention. The features of a protein or (poly)peptide are defined elsewhere in this application.

In a preferred embodiment, the protein of the invention comprises LacI.

In an alternative aspect, the present invention relates to a vector comprising the nucleic acid molecule of the invention. The properties of a vector have been described elsewhere in this application. In addition to the features already described, the vector of the present invention can be a prokaryotic vector. Prokaryotic vectors and their properties are well known in the art. Preferably, the vector is a plasmid, cosmid, virus, bacteriophage or another vector conventionally used e.g. in genetic engineering.

The nucleic acid molecule may be inserted into several commercially available vectors. Non-limiting examples include vectors compatible with an expression in mammalian cells like pREP (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, pIZD35, pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (Edge Biosystems) pTriEx-Hygro (Novagen), pCINeo (Promega), Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNA1, pSPORT1 (GIBCO BRL), pGEMHE (Promega) or pSVL and pMSG (Pharmacia, Uppsala, Sweden).

For vector modification techniques, see Sambrook and Russell (Molecular Cloning: A Laboratory Manual; Cold Spring Harbor, 2001). Generally, vectors can contain one or more origin of replication (ori) and inheritance systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes.

The nucleic acid molecules inserted in the vector can e.g. be synthesized by standard methods, or isolated from natural sources. Ligation of the coding sequences to transcriptional regulatory elements and/or to other amino acid encoding sequences can be carried out using established methods.

In a preferred embodiment of this aspect of the present invention, the vector further comprises a nucleic acid molecule encoding a fusion protein comprising (i) a fluorescent (poly)peptide, the excitation and/or emission wavelength of which differs from that of the fluorescent (poly)peptide comprised in the protein encoded by the nucleic acid molecule of the invention and (ii) a prey (poly)peptide.

In a different aspect, the present invention relates to a eukaryotic cell transfected with the vector of the invention.

It is preferred that the eukaryotic cell is a mammalian cell.

In a preferred embodiment of this aspect of the invention, the eukaryotic cell is transfected with the vector comprising the nucleic acid molecule of the invention and with a vector comprising a nucleic acid molecule encoding a fusion protein comprising (i) a fluorescent (poly)peptide, the excitation and/or emission wavelength of which differs from that of the fluorescent (poly)peptide comprised in the protein of the invention and (ii) a prey (poly)peptide.

The prey and the bait constructs of the invention can be comprised in only one vector, which might facilitate the comparison of expression levels.

In another aspect, the present invention relates to a eukaryotic cell having multiple copies of the lac-operator stably integrated in its DNA and stably expressing the protein of the invention.

In another aspect, the invention relates to a eukaryotic cell having multiple copies of the lac-operator stably integrated in its DNA and stably expressing a protein comprising (i) a (poly)peptide that binds to the lac operator; and (ii) a (poly)peptide specifically binding to GFP.

In a preferred embodiment, said (poly)peptide specifically binding to GFP comprises a (poly)peptide having the amino acid sequence of SEQ ID NOs: 7 or 9 or encoded by the nucleic acid sequence of SEQ ID NOs: 8 or 10.

The present invention also relates to a nucleic acid molecule encoding a fusion protein comprising (i) a (poly)peptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell; and (ii) a (poly)peptide specifically binding to GFP; as well as the protein encoded by said nucleic acid molecule, a vector comprising said nucleic acid molecule and a eukaryotic cell transfected with the vector comprising said nucleic acid molecule. The preferred embodiments and definitions as described for the aspects of the present invention relating to an F2H system comprising a bait and at least one prey construct are equally applicable to the above aspects of the invention utilizing the (poly)peptide specific for GFP. All embodiments relating to the first, second or third fusion protein of the first three aspects of the invention equally apply to the four fusion proteins of the aspects of the invention utilizing the (poly)peptide specific for GFP.

The figures show:

FIG. 1

Schematic outline of the fluorescent two-hybrid (F2H) assay. (a) Outline of pF2H-prey and pF2H-bait expression vectors coding for fluorescently labeled prey- and bait-proteins used for the F2H assay (b) The Lad domain of the bait-protein mediates binding to the chromosomally integrated lac operator array, which is visible as a fluorescent spot in nuclei of transfected cells. If the differentially labeled prey interacts with the bait it becomes enriched at the same spot resulting in co-localization of fluorescent signals at the lac operator (visible as yellow spot in the overlay image). (c) If the prey does not interact with the bait protein it remains dispersed in the nucleus and the lac operator array is only visualized by the bait protein (red spot). FP 1 and FP2 refer to two distinguishable fluorescent proteins, e.g. GFP or YFP and mCherry or mRFP.

FIG. 2

Specific interaction of DNA Ligase III with XRCC1 revealed by F2H (a) Transgenic BHK cells containing a chromosomal lac operator array were co-transfected with XRCC1-LacI-RFP and GFP-tagged DNA Ligase III or DNA Ligase I constructs. The lac repressor part of the XRCC1-LacI-RFP fusion protein mediates binding to the lac operator array (visible by fluorescence microscopy as red spot). DNA Ligase III is recruited to the lac operator array through interaction with XRCC1. Note that the highly homologous DNA Ligase I does not accumulate at the lac operator array indicating that it does not interact with XRCC1. Scale bars 5 μm. (b) Comparison of F2H results and co-immunoprecipitation (Co-IP) experiments. Co-IPs were performed with HEK 293T cells co-expressing RFP-XRCC1 and GFP-Ligase III or GFP-Ligase I, respectively. For interaction mapping the GFP-tagged BRCT domain of DNA Ligase III and a deletion construct lacking the BRCT domain were used. Immunoprecipitations were performed with a GFP-nanotrap (Rothbauer et al., 2007) (as shown before (Mortusewicz et al., 2006)). Precipitated fusion proteins were then detected with specific antibodies against RFP and GFP on western blots. RFP-XRCC1 was co-precipitated with GFP-Ligase III but not with GFP-Ligase I. RFP-XRCC1 was also co-precipitated with GFP-Ligase III BRCT but not with GFP-N-Ligase III BRCT. For comparison of F2H results the input (left) and bound (right) bands from Co-IPs were aligned with corresponding signals from the F2H assay. The Lad spot of the XRCC1-LacI-RFP bait construct shown in red and the bound fraction was aligned with the respective signal of the GFP-tagged prey constructs. Whole cell images of the respective F2H experiments are shown in (a) and FIG. 6.

FIG. 3

F2H analysis of cell cycle independent interaction of Dnmt1 with PCNA. (a) Schematic outline of full-length mouse Dnmt1 and fusion proteins. PBD, PCNA binding domain; NLS, nuclear localization sequence; TS, targeting sequence; ZnF, Zn²⁺-binding region; BAH 1 and 2, two Bromo Adjacent Homology domains. (b) Outline of binding possibilities of fusion proteins at the lac operator (lac op) array and at the replication fork. (c) Transgenic BHK cells containing a chromosomal lac operator array were co-transfected with PBD-LacI-YFP and RFP-PCNA constructs. RFP-PCNA shows the characteristic cell cycle dependent distribution (dispersed in non S phase cells (top row) and focal patterns in S phase (bottom row)). The lac repressor part of the PBD-LacI-YFP fusion protein mediates binding to the lac operator array (visible as green spot and highlighted by arrowheads) and the PBD mediates binding to PCNA at replication sites (focal pattern in S phase). Notice, RFP-PCNA is localized at the lac operator array in S and non S phase cells indicating an interaction of the PBD of Dnmt1 with PCNA throughout the cell cycle and independent of the replication machinery. (d) BHK cells were transfected with expression vectors for PBD-LacI-YFP and RFP-PCNA. As above, RFP-PCNA shows a disperse distribution in non S phase (top row) and redistributes to replication sites in S phase (bottom row). The PBD-LacI-YFP fusion protein binds to the lac operator array (green spot marked by arrowhead) but does not bind to replication sites in S phase since it lacks the PBD. Importantly, in these cells RFP-PCNA (prey) is not localized at the lac operator array (marked by arrowheads) indicating that binding depends on the presence of the PBD, which is absent in PBD-LacI-YFP (bait). Scale bars 5 μm

FIG. 4

Analysis of Huntington's disease related interactions by F2H. Reported interactions between (a) SUMO3 and HZFH and (b) HZFH and Vimentin revealed by F2H. (c) F2H analysis shows no interaction between SUMO3 and Vimentin as previously described (Goehler et al., 2004). In (b) the nucleus is outlined by a dashed line and in (c) the lac operator array is indicated (arrowheads). Scale bars 5 μm.

FIG. 5

Analysis of mitochondrial protein-protein interactions and the effect of a mutation associated with the Mohr-Tranebjaerg Syndrome. (a) Schematic overview of the hexameric DDP1-TIMM13 complex in the intermembrane space (IMS) of mitochondria. (b+c) BHK cells expressing the bait-protein mCherry-LacI-TIMM13 together either with GFP-DDP1 (b) or the loss-of-function mutant GFP-DDP1^(C66W) (c). While the functional wt fusion GFP-DDP1 shows interaction with TIMM13 revealed by co-localization of fluorescent signals at the lac operator array (b), the GFP-DDP1^(C66W) mutant is dispersedly distributed throughout the nucleus indicating no interaction (c). Scale bars 5 μm.

FIG. 6

BRCT mediated interaction of DNA-Ligase III with XRCC1 revealed by the F2H assay. Transgenic BHK cells containing a lac operator array were co-transfected with XRCC1-LacI-RFP and various GFP-tagged DNA-Ligase III constructs. The lac repressor part of the XRCC1-LacI-RFP fusion protein mediates binding to the lac operator array (visible as red spot). The BRCT domain is necessary and sufficient for targeting of DNA Ligase III to the lac operator array through interaction with XRCC1. Note that the highly homologous DNA Ligase I does not accumulate at the lac operator array indicating that it does not interact with XRCC1. Scale bars 5 μm.

FIG. 7

The F2H assay reveals the interaction of XRCC1 with PCNA, PARP-1 and PARP-2. BHK cells containing a lac operator array were transfected with expression vectors for XRCC1-LacI-RFP and either GFP-PARP-1, GFP-PARP-2 or GFP-PCNA. The lac repressor part of the XRCC1-LacI-RFP fusion protein mediates binding to the lac operator array (visible as red spot). GFP-PARP-1, GFP-PARP-2 and GFP-PCNA are targeted to the lac operator array indicating an interaction with XRCC1.Scale bar 5 μm.

FIG. 8

The F2H assay reveals the PBD-mediated interaction of DNA-Ligase I with PCNA. Transgenic U2OS cells containing a lac operator array were co-transfected with NLS-PCNA-LacI-RFP and various GFP-tagged DNA-Ligase I constructs. The lac repressor part of the NLS-PCNA-LacI-RFP fusion protein mediates binding to the lac operator array (visible as red spot). The PBD is necessary and sufficient for targeting of DNA Ligase I to the lac operator array through interaction with PCNA. Scale bar 5 μm.

FIG. 9

Interaction of various replication and repair proteins with PCNA revealed by the F2H assay. Transgenic BHK cells containing a lac operator array expressing NLS-PCNA-LacI-RFP and various GFP-tagged replication and repair proteins. All proteins tested interact with PCNA. Scale bar 5 μm.

The examples illustrate the invention.

EXAMPLE 1 Materials and Methods Expression Constructs

The Lad encoding sequence was PCR amplified from the p3′SS EGFP-LacI expression vector (Robinett et al., 1996) using the following primers: forward primer 5′-TCT AGA AAG CTT TCC ATG GTG AAA CCA GTA-3′ and reverse primer 5′-CCA TGC CCG GGA CAG GCT GCT TCG GGA AAC-3′ (restriction sites in italic). This PCR fragment was digested with HindIII and XmaI and cloned into the same sites of two Dnmt1-YFP expression vectors (MTNY.2 and PBHD-YFP) (Easwaran et al., 2004) generating PBD-LacI-YFP and PBD-LacI-YFP. The NLS-PCNA-LacI-RFP and XRCC1-LacI-RFP constructs were generated by PCR amplification of the PCNA and XRCC1 cDNA using the following primers (restriction sites in italic):

PCNA forward 5′-CCCCCTCGAGATGTTCGAGGCGCGC-3′ PCNA reverse 5′-GGGGAAGCTTGGAGATCCTTCTTCATCCTC-3′ XRCC1 forward 5′-CCCCAGATCTATGCCGGAGATCCGC-3′ XRCC1 reverse 5′-GGGGGAATTCGGGGCTTGCGGCACCAC-3′

Subsequently the PCR fragments were cloned into a LacI-RFP expression vector using the XhoI/HindIII sites for the NLS-PCNA-LacI-RFP and the BglII/EcoRI sites for the XRCC1-LacI-RFP expression vector.

All other F2H constructs were generated by PCR amplification of coding cDNAs and subsequent ligation into the AsiSI and NotI sites of the bait and prey expression vectors described in FIG. 1 a. The following primers were used with the restriction site indicated in italics:

DDP1 forward 5′-CCCCGCGATCGCGATTCCTCCTCCTCTTCCTC-3′ DDP1 reverse 5′-CCCCGCGGCCGCTCAGTCAGAAAGGCTTTCTG-3′ TIMM13 forward 5′-CCCCGCGATCGCGAGGGCGGCTTCGGCTCC-3′ TIMM13 reverse 5′-CCCCGCGATCGCGAGGGCGGCTTCGGCTCC-3′ HZFH forward 5′-GGGGGCGATCGCCACGCCCGCTTCC-3′ HZFH reverse 5′-CCCCGCGGCCGCTTAGTCGTCTATACAGATCACCTCC-3′ SUMO3 forward 5′-CCCCGCGATCGCGCCGACGAAAAGCCCAAG-3′ SUMO3 reverse 5′-CCCCGCGGCCGCTCAGTAGACACCTCCCG-3′ Vim forward 5′-GGGGTGTACAGCGATCGCATGTCGACCCACGCGT-3′ Vim reverse 5′-CCCCGAATTCGCGGCCGCTTATTCAAGGTCATCGTGATGCT-3′

Mammalian expression constructs encoding translational fusions of human DNMT1, DNA-Ligase I, DNA-Ligase III, p21, FEN I, Polymerase p66 subunit, PARP-1, PARP-2 and PCNA were previously described (Cazzalini et al., 2003; Maeda et al., 2006; Meder et al., 2005; Mortusewicz et al., 2005; Schermelleh et al., 2005; Sporbert et al., 2005). Deletion constructs and isolated domains of DNA-Ligase I and III were described in Mortusewicz et al (Mortusewicz et al., 2006) Immunoprecipitations were performed with a GFP-nanotrap (Rothbauer et al., 2007) as described before (Mortusewicz et al., 2006). All fusions constructs were tested for correct expression and localization.

Cell Culture and Transfection

Transgenic BHK cells (clone #2) and U2OS cells (clone 2-6-3) containing lac operator repeats were cultured under selective conditions in DMEM supplemented with 10% fetal calf serum and 150 g/ml hygromycin B (PAA Laboratories) as described (Janicki et al., 2004; Tsukamoto et al., 2000). For microscopy cells were grown to 50-70% confluence either on 18×18 glass coverslips or in μ-slides (ibidi, Munich, Germany) and then co-transfected with the indicated expression constructs using Polyplus transfection reagent jetPEI™ (BIOMOL GmbH, Hamburg, Germany) according to the manufacturer's instructions. After 6-10 hours the transfection medium was changed to fresh culture medium and cells were then incubated for another 12-24 hours before live cell microscopy or fixation with 3.7% formaldehyde in PBS for 10 min at room temperature. Fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 3 min, counterstained with DAPI and mounted in Vectashield (Vector Laboratories, CA, USA).

Microscopy

Live or fixed cells expressing fluorescent proteins were analyzed using a Leica TCS SP2 AOBS confocal microscope equipped with a 63×/1.4 NA Plan-Apochromat oil immersion objective. Fluorophores were excited with a 405 nm Diode laser, a 488 nm and a 514 nm argon laser and a 561 nm Diode-Pumped Solid-State (DPSS) laser. Confocal image stacks of living or fixed cells were typically recorded with a frame size of 512×512 pixels, a pixel size of 50-100 nm, a z-step size of 250 nm and the pinhole opened to 1 Airy unit. A maximum intensity projection of several mid z-sections was generated with ImageJ (Version 1.38, http://rsb.info.nih.gov/ij/).

EXAMPLE 2 Method to Detect the Interaction of Proteins

To visualize protein-protein interactions in living cells in real time we developed a fluorescence two-hybrid (F2H) assay. The rationale for the F2H assay is based on the fact that proteins are freely roaming the cell unless interactions with other cellular components immobilize them at specific structures (Phair and Misteli, 2000).

We used a previously described BHK and an U2OS cell line which both harbor a stable integration of about 200-1000 copies of a plasmid carrying 256 copies of the lac operator sequence (Janicki et al., 2004; Tsukamoto et al., 2000). We generated an expression construct encoding a fluorescent bait protein consisting of a fluorescent protein (FP), the lac repressor (Lad) and the protein X to be tested for interactions (bait) resulting in the triple fusion protein FP-LacI-X (FIG. 1 a) or X-LacI-FP. This fusion protein binds to the operator array, which then becomes visible due to the focal enrichment of the FP signal. A second, differently labeled fusion protein (FP-Y, prey) may either interact with the bait protein X leading to co-localization of the FP signals (FIG. 1 b) or may not interact, resulting in a dispersed distribution of the prey fluorescence (FIG. 1 c).

EXAMPLE 3 Visualization of Interactions Between DNA Repair Proteins

To test the F2H assay, the previously described interaction between the two DNA repair proteins DNA Ligase III and XRCC1 (Caldecott et al., 1994; Wei et al., 1995) was analyzed and the results were compared with data obtained from pull down assays. We have previously shown that this interaction is mediated by the BRCT domain of DNA Ligase III which targets it to DNA repair sites (Mortusewicz et al., 2006). We generated a bait fusion protein consisting of XRCC1 followed by the Lad and the monomeric red fluorescent protein RFP (mRFP). As expected this fusion protein localized at the lac operator array in transiently transfected BHK cells (FIG. 2 a). Both, the full length GFP-tagged DNA Ligase III as well as the isolated GFP-labeled BRCT domain co-localize with XRCC1 at the lac operator array, while a fusion protein missing the BRCT domain shows a dispersed distribution. Notably, the highly homologous DNA Ligase I, which catalyzes the same reaction as DNA Ligase III, does not bind to XRCC1 (FIG. 2 a and FIG. 6). A direct comparison of the F2H data with data obtained from Co-IP experiments reveals that these two methods gave similar results (FIG. 2 b). In addition, we could also observe the recently described interaction of XRCC1 with PCNA (Fan et al., 2004) and the two DNA-damage dependent PARPs, PARP-1 and PARP-2 (Masson et al., 1998; Schreiber et al., 2002) (FIG. 7). These results demonstrate that the F2H assay is well suited to study protein-protein interactions in living cells.

EXAMPLE 4 Analysis of Cell Cycle Dependence of Protein-Protein Interactions

A challenge in the analysis of protein-protein interactions is to monitor transient changes caused by for example cell cycle progression or other external stimuli. We analyzed the previously described interaction between DNA methyltransferase 1 (Dnmt1) and PCNA which is mediated by the PCNA binding domain (PBD) and targets Dnmt1 to sites of DNA replication in S phase (Chuang et al., 1997; Easwaran et al., 2004). These findings raised the question whether this interaction occurs only in S phase at replication foci or throughout the cell cycle. We generated two bait-proteins comprising parts of Dnmt1 fused to the Lad and YFP. One bait (PBD-LacI-YFP) comprises aa 118-427 of Dnmt1 including the PBD, while the second bait (ΔPBD-LacI-YFP) lacks the PBD and comprises aa 629-1089 of Dnmt1 (FIG. 3 a). As a prey-protein we used RFP-PCNA which in addition marks sites of DNA replication allowing the identification of cells in S phase (Easwaran et al., 2005; Sporbert et al., 2005). The binding possibilities of these fusion proteins at the lac operator array and the replication fork are summarized in FIG. 3 b.

In non S phase the Lad part of the bait proteins only binds to the chromosomally integrated lac operator array, which—dependent on the ploidy of the cell—becomes visible as one or two fluorescent spots in the nucleus. Interaction of RFP-PCNA with the PBD part of the bait protein results in co-localization of the fluorescent signals at the lac operator array (FIG. 3 c upper panel), while deletion of the PBD in the bait protein leads to a dispersed distribution of RFP-PCNA in non S phase cells (FIG. 3 d upper panel). This clearly illustrates that the PBD-dependent interaction of Dnmt1 with PCNA also occurs outside of S phase. In S phase cells, RFP-PCNA localizes at sites of ongoing DNA replication and in addition is recruited to the lac operator array by the PBD-LacI-YFP bait protein (FIG. 3 c lower panel). In contrast, when RFP-PCNA is coexpressed together with a bait protein lacking a functional PBD (ΔPBD-LacI-YFP), RFP-PCNA is exclusively enriched at DNA replication sites and not at the lac operator array highlighted by ΔPBD-LacI-YFP (FIG. 3 d lower panel). These results clearly show that the localization of RFP-PCNA (prey) at the lac operator array depends on the presence of the PBD in the bait construct and that this interaction is not restricted to S phase.

Next we analyzed the interaction of other PBD-containing proteins with PCNA. We generated a bait fusion protein comprising PCNA fused to an additional NLS followed by Lad and RFP (NLS-PCNA-LacI-RFP). When co-expressed with GFP-Ligase I, both fusion proteins localized to the lac operator array indicating interaction between PCNA and DNA Ligase I. Deletion of the PBD lead to a disperse distribution of DNA Ligase I, while the PBD of DNA Ligase I alone was sufficient for binding to PCNA at the lac operator array (FIG. 8). This is in agreement with previous studies showing that the PBD of DNA Ligase I is necessary and sufficient for its targeting to DNA replication and repair sites (Cardoso et al., 1997; Montecucco et al., 1995; Mortusewicz et al., 2006). Notably, using the F2H assay we could demonstrate that DNA Ligase I, as well as the isolated PBD are capable of binding to PCNA also outside of S-phase. Likewise we could show binding of various additional replication and repair proteins like FEN1, p21 and the Polymerase subunit p66 to PCNA in non S-phase cells (FIG. 9). Taken together we could show that the interaction between replication proteins and PCNA is not limited to S phase but also occurs in non S phase cells and outside the replication machinery. This illustrates that the F2H assay offers the unique potential to analyze cell cycle specific changes in protein-protein interactions in living cells.

EXAMPLE 5 Detection of Interactions Between Proteins Related to Huntington's Disease

To investigate whether the F2H assay can also detect protein-protein interactions taking place in other cellular compartments, we tested the F2H assay with protein interactions identified in the context of Huntington's disease by yeast two-hybrid (Y2H) assays (Goehler et al., 2004). We analyzed the interaction of one cytoplasmatic (Vimentin) and two nuclear (HZFH and SUMO3) proteins. Vimentin has been described to be a cytoskeleton component and participates in transport processes, whereas HZFH and SUMO3 are involved in transcriptional regulation and DNA maintenance (Goehler et al., 2004). These proteins were either fused with a red fluorescent mCherry-LacI-NLS or with NLS-GFP to generate sets of bait and prey proteins. BHK cells carrying a lac operator array were transfected with all possible combinations of expression constructs and subjected to microscopic analysis. We could detect an interaction between Vimentin and HZFH independent of whether these two proteins were used as bait or prey (FIG. 4 and data not shown). We could also detect the reported interaction between SUMO3 and HZFH while Vimentin and SUMO3 did not interact, as previously described (FIG. 4) (Goehler et al., 2004). These results show that interactions of nuclear and cytoplasmic proteins can be studied with the F2H assay.

EXAMPLE 6 Detection of Interactions Between Mitochondrial Proteins

Next, we investigated whether the F2H assay is also suitable to detect protein-protein interactions occurring in other cellular organelles. To this end, we analyzed the interaction between two mitochondrial proteins, DDP1 (deafness dystonia peptide 1) and TIMM13.

Both proteins are nuclear encoded and imported into the mitochondrial intermembrane space (IMS) forming a hexameric complex (FIG. 5 a). Within the IMS the DDP1-TIMM13 complex facilitates the import of hydrophobic proteins of the mitochondrial import machinery into the mitochondrial innermembrane (Rothbauer et al., 2001). A mutation of the DDP1 gene was associated with the Mohr-Tranebjaerg-Syndrome, which is a progressive, neurodegenerative disorder (Tranebjaerg et al., 1995). This C66W missense mutation is known to cause a full blown phenotype and affects the highly conserved Cys(4) motif of DDP1. Previous studies have shown, that this amino acid exchange abolishes the interaction between DDP1 and TIMM13 in the IMS (Hofmann et al., 2002).

Using a red fluorescent bait fusion protein comprising LacI-NLS-TIMM13 and GFP-tagged wildtype (GFP-DDP1) or mutant DDP1 (GFP-DDP1^(C66W)) prey proteins we analyzed this specific mitochondrial protein interaction with the F2H assay. We found that GFP-DDP1 co-localizes with TIMM13 at the lac operator array (FIG. 5 b), while GFP-DDP1^(C66W) was evenly distributed (FIG. 5 c). These results demonstrate that the F2H assay is also suitable for the analysis of protein-protein interactions occurring outside the nucleus and the characterization of disease related point mutations disrupting these interactions.

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1. An in vitro method for detecting protein-protein interactions comprising: (a) expressing in an engineered eukaryotic cell a first fusion protein comprising three different polypeptides, wherein said three different polypeptides comprise: (i) a fluorescent polypeptide; (ii) a polypeptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell; and (iii) a bait polypeptide; wherein said engineered eukaryotic cell comprises the stable integration of a nuclear structure capable of binding the first fusion protein; and (b) expressing in the same engineered eukaryotic cell a second fusion protein comprising (i) a fluorescent polypeptide, the excitation and/or emission wavelength of which differs from that of the fluorescent polypeptide comprised in said first fusion protein; and (ii) a prey polypeptide to form a fluorescent prey polypeptide, wherein said fluorescent prey polypeptide does not, by itself, accumulate at distinct sites in the nucleus of the engineered eukaryotic cell; and (c) detecting the fluorescence emission of the fluorescent parts of the first and the second fusion protein in the engineered eukaryotic cell upon excitation, wherein a co-localization of the fluorescence emission of both fusion proteins at distinct sites in the engineered eukaryotic cell nucleus is indicative of an interaction of the bait and the prey polypeptide.
 2. An in vitro method for identifying a compound modulating the interaction of two polypeptides comprising (a) expressing in an engineered eukaryotic cell a first fusion protein comprising three different polypeptides, wherein said three different polypeptides comprise (i) a fluorescent polypeptide; (ii) a polypeptide that, when expressed in a cell, accumulates at distinct sites in the nucleus of the cell; and (iii) a bait polypeptide; and (b) expressing in the same engineered eukaryotic cell a second fusion protein comprising (i) a fluorescent polypeptide, the excitation and/or emission wavelength of which differs from that of the fluorescent polypeptide comprised in said first fusion protein; and (ii) a prey polypeptide known or suspected to interact with the bait polypeptide to form a fluorescent prey polypeptide, wherein said fluorescent prey polypeptide does not, by itself, accumulate at distinct sites in the nucleus of the engineered eukaryotic cell; (c) contacting the engineered eukaryotic cell with a test compound; and (d) detecting the fluorescence emission of the fluorescent parts of the first and the second fusion protein in the cell upon excitation; wherein said engineered eukaryotic cell comprises the stable integration of a nuclear structure capable of binding the first fusion protein; and wherein a change in the degree of co-localization of the fluorescence emission of the fluorescent parts of both fusion proteins at distinct sites in the engineered eukaryotic cell nucleus as compared to that observed in the nucleus of an engineered reference cell not contacted with the test compound is indicative that the compound is capable of modulating the interaction of the bait and the prey polypeptide.
 3. The method of claim 2, wherein a decrease of co-localization of the fluorescent parts of both fusion proteins in the engineered eukaryotic cell nucleus as compared to that observed in the nucleus of an engineered reference cell not contacted with the test compound is indicative of the compound being capable of weakening the interaction of the bait and the prey polypeptide.
 4. The method of claim 2, wherein an increase in co-localization of the fluorescent parts of both fusion proteins in the engineered eukaryotic cell nucleus as compared to that observed in the nucleus of an engineered reference cell not contacted with the test compound is indicative of the compound being capable of inducing or enhancing the interaction of the bait and the prey polypeptide.
 5. The method of claim 1, further comprising: (d) monitoring the fluorescence emission of the fluorescent parts of the first and the second fusion protein in the engineered eukaryotic cell in the course of one or more processes in the engineered eukaryotic cell; wherein a change in the degree of co-localization of the fluorescence emission of the fluorescent parts of both fusion proteins in the engineered eukaryotic cell is indicative of a dependency of the interaction on the one or more cellular processes.
 6. The method of claim 5, wherein the cellular process is the cell cycle, secretion, translocation, signal transduction or DNA repair.
 7. The method of claim 1, wherein a co-localization of the fluorescence emission of the fluorescent parts of both fusion proteins is detected, further comprising selective extinction of the fluorescence of said second fusion protein and monitoring the restoration of co-localization of the fluorescence emission of the fluorescent parts of both fusion proteins over time, wherein the time needed to establish co-localization is indicative of the strength of the protein-protein interaction.
 8. The method of claim 1, wherein components (i), (ii) and/or (iii) of said first fusion protein and/or components (i) and (ii) of said second fusion protein are connected via a linker.
 9. The method of claim 1 wherein the polypeptide that, when expressed in an engineered cell, accumulates at distinct sites in the nucleus of the cell directly interacts with proteinaceous or non-proteinaceous structures accumulated at distinct sites in the nucleus of the engineered eukaryotic cell.
 10. The method of claim 1 wherein the polypeptide that, when expressed in an engineered eukaryotic cell, accumulates at distinct sites in the nucleus of the cell indirectly interacts with proteinaceous or non-proteinaceous structures accumulated at distinct sites in the nucleus of the engineered eukaryotic cell.
 11. The method of claim 2, wherein components (i), (ii) and/or (iii) of said first fusion protein and/or components (i) and (ii) of said second fusion protein are connected via a linker.
 12. The method of claim 1 wherein any of the prey polypeptides comprises a nuclear localization signal.
 13. The method of claim 1 wherein expression in the engineered eukaryotic cell is effected by transfecting the nucleic acid molecules encoding said first and second fusion protein in one or more vectors.
 14. The method of claim 1 wherein the distinct sites of the engineered eukaryotic cell form inert structures.
 15. The method of claim 14, wherein the inert structure is the nuclear lamina or nuclear speckles.
 16. The method of claim 1 wherein the polypeptide that, when expressed in an engineered eukaryotic cell, accumulates at distinct sites in the nucleus of the engineered eukaryotic cell interacts with a member of the group selected from DNA and PML bodies.
 17. The method of claim 1 wherein the polypeptide accumulated at distinct sites of the nucleus is heterologous to the engineered eukaryotic cell.
 18. The method of claim 16 wherein the interaction is with DNA and the DNA is the lac-operator.
 19. The method of claim 18, wherein the lac-operator is present in the nucleus of the engineered eukaryotic cell in multiple copies.
 20. The method of claim 1 wherein said first fusion protein comprises the lac repressor. 